Antiviral effects and tissue exposure of tetrandrine against SARS‐CoV‐2 infection and COVID‐19

Abstract Tetrandrine (TET) has been used to treat silicosis in China for decades. The aim of this study was to facilitate rational repurposing of TET against SARS‐CoV‐2 infection. In this study, we confirmed that TET exhibited antiviral potency against SARS‐CoV‐2 in the African green monkey kidney (Vero E6), human hepatocarcinoma (Huh7), and human lung adenocarcinoma epithelial (Calu‐3) cell lines. TET functioned during the early‐entry stage of SARS‐CoV‐2 and impeded intracellular trafficking of the virus from early endosomes to endolysosomes. An in vivo study that used adenovirus (AdV) 5‐human angiotensin‐converting enzyme 2 (hACE2)‐transduced mice showed that although TET did not reduce pulmonary viral load, it significantly alleviated pathological damage in SARS‐CoV‐2‐infected murine lungs. The systemic preclinical pharmacokinetics were investigated based on in vivo and in vitro models, and the route‐dependent biodistribution of TET was explored. TET had a large volume of distribution, which contributed to its high tissue accumulation. Inhaled administration helped TET target the lung and reduced its exposure to other tissues, which mitigated its off‐target toxicity. Based on the available human pharmacokinetic data, it appeared feasible to achieve an unbound TET 90% maximal effective concentration (EC90) in human lungs. This study provides insights into the route‐dependent pulmonary biodistribution of TET associated with its efficacy.


INTRODUCTION
The COVID-19 pandemic is a public health concern worldwide. Although several small-molecule drugs, including remdesivir, molnupiravir, nirmatrelvir, and ritonavir have been formally approved or granted emergency use authorization for the treatment of COVID-19, SARS-CoV-2-specific therapeutics that can prevent severe illness, hospitalization, and mortality are still in great demand.
Tetrandrine (TET) and cepharanthine are bisbenzylisoquinoline alkaloids extracted from herbal plants. Multiple studies have suggested that both compounds have antiinflammatory and immunoregulatory properties. 1 Cepharanthine and TET have antiviral activity against human coronavirus OC43 (HCoV-OC43) and SARS-CoV-2 in Vero cell screens. 2 TET is a potent blocker of two-pore iron channels (TPCs), which are located in the membranes of host endolysosomes (ELs) and are critical for viral entry. TET can inhibit infection by various viruses, including herpes simplex virus, dengue virus, Ebola virus, Middle East respiratory syndrome coronavirus (MERS-CoV), and HCoV-OC43. [3][4][5] Additionally, TET has a broad spectrum of pharmacological activities, including anti-cancer, anti-inflammatory, immunosuppressant, anti-fibrotic, and anti-arthritic properties. 6 Because the disease severity of COVID-19 is linked to an excessive inflammatory response and pulmonary fibrosis, 7 TET may potentially be used in the treatment of COVID-19 due to its anti-inflammatory and immunomodulatory effects. As one of the most promising antiviral candidates, 8 TET has been reported to have a significant inhibitory effect against SARS-CoV-2 pseudotyped virus and authentic virus infection in Vero E6 cells. 5,8 However, the in vivo efficacy of TET in inhibiting SARS-CoV-2 replication and curing disease remains unclear.
In vivo exposure is the most important factor that affects drug efficacy. A 90%-99% reduction in viral replication is thought to be required for effective therapy of rapidly progressive acute viral infection. 9 For example, the concentration required for a 90% reduction in SARS-CoV-2 virus replication (90% maximal effective concentration, EC 90 ) is usually obtained using physiologically relevant cellular systems in the first phase. 9 Although the significant role of EC 90 in evaluating in vivo antiviral efficacy is well accepted, how to correctly compare the in vivo concentration with in vitro EC 90 values warrants in-depth investigation. Due to the inaccessibility of tissue concentration, plasma concentration is generally used as a surrogate for the concentration at the main site of action of the disease. 10 In this study, the in vitro and in vivo anti-SARS-CoV-2 activities of TET were evaluated, and the tissue exposure characteristics of TET in rats administered by different routes as single and multiple doses were determined. Particular attention was given to target exposure to TET in the lung tissue. The anti-SARS-CoV-2 EC 90 was correlated with unbound lung concentration. Based on the human pharmacokinetics (PK) of TET available in the literature, we highlighted the potential of TET for the treatment of COVID-19.

Anti-SARS-CoV-2 activity of tetrandrine in vitro
The anti-SARS-CoV-2 activity of TET was evaluated in three cell lines, including African green monkey kidney Vero E6 cells, human hepatocarcinoma cell line (Huh7), and human lung adenocarcinoma epithelial cells (Calu-3). Firstly, the cytotoxicity of the drug in these cell lines was measured by cell-counting kit-8 (CCK8) assay. Then, the cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of either 0.05 or 0.01 in the presence of TET or dimethyl sulfoxide (DMSO) as control. The doseresponse curves were determined by detection of viral RNA levels by quantitative real-time polymerase chain reaction (qRT-PCR) in the supernatant of the infected cell at 24 or 48 h post-infection (p.i.). As shown in the upper panel of Figure 1, TET efficiently inhibited SARS-CoV-2 infection (MOI = 0.05) in Vero E6 cells, with a 50% cytotoxic concentration (CC 50 ) and 50% maximal effective concentration (EC 50 ) of 24.51 and 2.36 µM, respectively, and a selectivity index (SI = CC 50 /EC 50  . The viral yield in the supernatant was quantified by quantitative real-time polymerase chain reaction (qRT-PCR). CC 50 , 50% cytotoxic concentration; EC 50 , 50% maximal effective concentration; SI, selectivity index (SI = CC 50 /EC 50 ) in Vero E6, Huh7, and Calu-3 cells were 1.85, 1.25, and 1.60 µM, respectively. These results indicate that TET has direct antiviral activity against SARS-CoV-2 infection in vitro.

Tetrandrine functions through trapping virions within intracellular early endosomes
As shown in Figure 2, incubation with TET during virus entry and post-entry processes potently inhibited virus production. TET was more effective for inhibiting production on virus entry (approximately 90% inhibition rate) than on the post-entry process (approximately 70% inhibition rate) (Figure 2A). In addition, western blotting ( Figure 2B) and immunofluorescence analysis (IFA) ( Figure 2C) showed that the expression level of viral nucleocapsid protein (NP) was more markedly reduced at the entry stage than at the post-entry stage.
We further explored the mechanism of TET action as an inhibitor of endolysosomal ion channels in inhibiting viral entry by analyzing viral intracellular trafficking ( Figure 3). Colocalization analysis ( Figure 3A) of virions with early endosomes (EEs) or ELs showed that at 15 min p.i., the amounts of internalized virions located in early endosome antigen 1 (EEA1 + ) EEs were similar in the DMSO-and TET-treated groups (24.5% and 24.1%, respectively). As the time of infection increased, the number of virions that colocalized with EEs decreased significantly in the DMSO group (21.1% and 6.7% at 30 and 45 min p.i., respectively), whereas increased internalized virions were observed in lysosome-associated membrane glycoprotein (LAMP) + ELs (17.5% and 22.6% at 45 and 60 min p.i., respectively), indicating that some virions had already been transported from EEs to ELs. In contrast, in the presence of TET, more virions were detected in the EEs (28.0% at 30 min and 21.6% at 45 min; p < 0.001), and less virions were located in the ELs (7.7% at 45 min and 7.3% at 60 min; p < 0.001), suggesting that TET trapped the virions in the EEs. In addition, TET treatment caused an enlargement and discontinuity of the membrane of the EE and EL vesicles ( Figure 3B). Considered together, these results suggest that TET impedes intracellular trafficking of SARS-CoV-2 in endosomes.

Tetrandrine deceases the pathological lesions caused by SARS-CoV-2 infection in vivo
Adenovirus (AdV) 5-hACE2-transduced type I interferon receptor knockout (IFNAR −/− ) mice were used to F I G U R E 2 Time-of-addition assay of tetrandrine (TET). SARS-CoV-2-infected Vero E6 cells were treated with TET at "full-time", "entry", or "post-entry" process. Virus yield in the infected cell supernatants was quantified by quantitative real-time polymerase chain reaction (qRT-PCR) (A) and nucleocapsid protein (NP) expression in infected cells was analyzed by western blotting (B) and immunofluorescence assay (C) at 16 h post-infection (p.i.). Bar = 200 µm. ns indicates p > 0.05 and **p < 0.01 evaluate the therapeutic potential of TET against SARS-CoV-2 infection in vivo. This infection model has been widely used for evaluation of SARS-CoV-2 antivirals [11][12][13][14] ( Figure 4A). Neither the viral RNA levels ( Figure 4B, left) nor the infectious viral yields ( Figure 4B, right) in the lungs decreased significantly after TET treatment at any of the three drug doses tested. However, histopathology analysis (hematoxylin and eosin staining) showed that TET alleviated pathological lesions in the lungs of SARS-CoV-2-infected mice. As shown in Figure 4C, at 5 days p.i., the lungs of the control mice displayed severe diffuse alveolar damage (DAD), with marked thickening of alveolar septa, extensive exudation of protein in the alveoli, obvious epithelial hyperplasia in the bronchi and bronchioles, and extensive immune infiltration in the alveoli and around the bronchi and vessels. In contrast, in the mice treated with 90 or 60 mg/kg of TET, only slight lung damage was observed at 5 days p.i., as evidenced by the intact alveolar septa and cavities, and small numbers of immune cell infiltrate around the bronchi/bronchioles and blood vessels. In the low-dose group (30 mg/kg), mild/moderate levels of DAD and interstitial inflammation were observed ( Figure 4C).

2.4
Pharmacokinetic behavior of tetrandrine in rats TET alleviated lung pathology without decreasing the viral load, suggesting that it would be useful to assess the PK properties of TET in vivo and explore the optimal Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) methods of measuring TET in rat plasma and tissue homogenates were validated for selectivity, accuracy, precision, matrix effect, recovery, and stability. Calibration curves ranged from 2 to 2000 ng/ml in plasma and 5 to 2000 ng/ml in tissue homogenates. The intra-day and inter-day accuracy and precision met the standards for four different quality control concentrations. The relative standard deviations of the matrix effects were within 11%. The results showed that TET was stable in the matrix under various storage conditions (detailed bioanalysis methods are available in Supporting Information; the corresponding results are shown in Table S1-S3 and Figure S2).
The mean plasma concentration-time curves after single intravenous (i.v., 5 mg/kg), oral (per os [p.o.], 30 mg/kg), and intratracheal (i.t., 7 and 14 mg/kg) administration are shown in Figure 5. There was a significant difference in plasma concentrations between male and female rats, regardless of the dosing route. The plasma concentration in female rats was much higher than that in male rats. The major PK parameters of male rats are presented in Table S4. TET had low plasma clearance (CL, 13.6 ml/min/kg) and a large volume of distribution (V d , 24.8 L/kg) in male rats. Following oral administration, TET was absorbed slowly (T max = 6.7 h), with moderate bioavailability (F = 33.6%) in rats. Compared to the fraction of absorbed (F a × F g ) (36.5%), hepatic metabolism played a minor role in the oral first-pass effect on TET. TET was rapidly absorbed (T max = 0.083-0.139 h) via i.t. administration compared to p.o. administration. The plasma concentration declined quickly after i.v. and i.t. administration in the distribution phase, but decreased smoothly in the elimination phase, indicating that TET is distributed rapidly to tissues and released slowly from tissues into the blood.

Route-dependent tissue distribution of TET in rats
The mean exposures of TET in various tissues after oral (single and repeated at 30 mg/kg) and i.t. (7 mg/kg) administration are shown in Figure 6. Taking tissue protein binding rates into consideration (Table S5), it was found that the biodistribution of TET in tissues showed three characteristics (Tables 1 and S6-S8). First, the exposure  in tissues was much higher than that in plasma, with unbound tissue partition ratio (K p ) values >1, which is consistent with the large volume of distribution (V d = 24 L/kg) obtained after i.v. administration. Second, the amount of exposure in various tissues could be sorted into three tiers: high level with K p,uu > 20 (including the lung, spleen, liver, and kidney), moderate level with 2 < K p,uu < 10 (including the stomach, muscle, heart, and testis), and low level with K p,uu < 2 (including the brain, intestine, and adipose tissue). Third, the rank orders of TET biodistribution were similar in the three dosing patterns, with higher accumulation in the lung in each case. However, inhalation of TET led to the much higher exposure in the lung and relatively lower liver exposure compared to oral administration. Although high lung exposure to TET is the desired outcome, TET also causes hepatic toxicity. However, our results showed that inhalation could improve lung exposure to TET at lower dose levels, which might achieve a local effect without side effects.

In vitro metabolic stability and CYP reaction phenotyping
The metabolic stability of TET (1 µM) was measured in rat liver microsomes (RLM) and human liver microsomes (HLM) fortified with nicotinamide adenine dinucleotide phosphate (NADPH) using the substrate depletion method (Table S9). Intrinsic clearances (CL int ) of TET in RLM (CL int = 61.5 ml/min/mg protein) and HLM (CL int = 46.7 ml/min/mg protein) were obtained. Hepatic blood clearance (CL h,blood ) was calculated from CL int using physiological constants from rats and humans, unbound fractions, and blood/plasma partitioning (R b/p , 3.08, measured in rat blood and used as a surrogate for humans). The extrapolated CL h values for rats (CL h = 1.8 ml/min/kg) and humans (CL h = 0.628 ml/min/kg) were very low.
The metabolic turnover of TET was also measured using a panel of recombinant P450s. As shown in Figure S3A, incubation of TET with human CYP3A4 and CYP3A5 led to depletion of the parent drug. According to the total normalized rate (TNR) approach, the metabolic contributions of CYP3A4 and CYP3A5 were 88.3% and 11.7%, respectively.

Correlation between unbound tetrandrine concentration and 90% maximal effective concentration
Given that only unbound drugs could exert a therapeutic effect by crossing the membrane barrier, the unbound EC 90 obtained in Vero E6, Huh7, and Calu-3 cells, and TET pulmonary concentrations were measured to estimate whether unbound TET in the lungs is effective against SARS-CoV-2. As the free fraction of TET was 0.202 in the culture medium (Table S5), the exact unbound EC 90 values were 1.55 µM (0.96 µg/ml), 1.57 µM (0.97 µg/ml), and 2.44 µM (1.52 µg/ml) in Vero E6, Huh7, and Calu-3 cells, respectively. The unbound EC 90 and unbound TET concentrations in the lungs after p.o. and i.t. administration are compared in Figure 7. The unbound TET in the lungs after a single oral dose of 30 mg/kg was below the unbound EC 90 threshold ( Figure 7A). However, administering repeated oral doses of 30 mg/kg led to unbound TET levels in the lungs, considerably above the EC 90 , which was maintained for at least 96 h ( Figure 7B). Compared with oral administration, the unbound TET concentration in the lungs was close to the EC 90 , and the TET level in other tissues was lower after i.t. administration at 7 mg/kg than after oral dosing. Therefore, it may be possible to achieve the efficacious EC 90 in the lungs after i.t. administration, if the dosage, frequency, and interval are optimized.

DISCUSSION
To our knowledge, this is the first study to show that TET has anti-SARS-CoV-2 potency in vitro and significantly alleviates murine lung injuries induced by SARS-CoV-2 infection. Although TET has been widely used to treat silicosis in China for nearly 50 years, 15 there are few studies available on its systematic PK. The unavailability of data makes it difficult to bridge in vivo PK data with in vitro efficacy results according to the standard drug repositioning approach. In this study, plasma PK and tissue distribution results from different routes of administration in rats and data on in vitro protein binding fraction, metabolic stability, and therapeutic efficacy were integrated to measure the exposure-response relationships and identify the optimal administration route of TET to achieve preferential biodistribution and effective lung targeting. Finally, combined with the available human PK data, 16 the clinical potential of TET for the treatment of SARS-CoV-2 infection was evaluated.
As a known blocker of the TPC, TET appears to have broad antiviral activity. It can inhibit the host cell entrance of the Ebola virus with an EC 50 of 55 nM, HCoV-OC43 with an EC 50 Figure 1A,B). The EC 50 and EC 90 of TET against SARS-CoV-2 are higher (10.37 and 14.80 µM) in Vero E6 cells overexpressing transmembrane protease serine 2 (TMPRSS2, a co-receptor for SARS-CoV-2 entry). 18 The inhibition potency of TET was less in Calu-3 lung cells (EC 50 of 5.03 µM and EC 90 of 12.08 µM) than in Vero E6 and Huh7 cells at MOI of 0.05 ( Figure 1A-C). This may be due to the high expression levels of TMPRSS2 in Calu-3 cells, leading to a partial bypass of virus entry via the endocytosis pathway. 19 However, the EC 50 values of TET against  Figure 1D-F). Thus, the underlying mechanism causing the difference remains to be further investigated.
Although, roughly compared with the currently approved clinical drugs, such as remdesivir (EC 50 ≈ 0.7 µM) and molnupiravir (EC 50 ≈ 0.3 µM), both of which directly bind to viral proteins, 20,21 the EC 50 value of TET against SARS-CoV-2 was higher, TET targets host TPC1 and TPC2 and may have different antiviral mechanisms. TPC1 and TPC2 are located within the endolysosomal system, where they regulate the endolysosomal microenvironment and trafficking dynamics. 19,22 TPC blockers or genetic ablation of TPCs affects endosomal trafficking of viruses and bacterial toxins, reducing infectivity. 17,23 TPC activity alters endolysosomal Ca 2+ content and pH, which could impair endolysosomal trafficking and the activity of proprotein convertases (such as furin) required for proteolytic activation of the spike protein in coronavirus fusion. 17,24 Ou et al. 5 showed that inhibition of TPC2 blocks SARS-CoV-2 pseudovirion entry. Here, we also showed that TET mainly inhibited SARS-CoV-2 at the early-entry stage by dysregulating virus trafficking among the ELs and trapping the viral particles within the intracellular EEs in Vero E6 cells (Figures 2 and 3). In addition, consistent with previous reports, 17,25 we observed that TET enlarged EEs and ELs and disrupted the regular vesicle membrane (Figure 3), which may also affect its maturation and functionality.
Further in vivo studies using the AdV5-hACE2transduced mouse model showed that TET did not reduce pulmonary viral load but significantly reduced virusinduced murine lung injury in a dose-dependent manner (Figure 4). In severe COVID-19, DAD is prominent, with fibrin exudates, hyaline membrane formation, thickening of the alveolar walls, and an uncontrolled inflammatory response, and up to 17% of individuals with COVID-19 develop pulmonary fibrosis. 7 TET is an anti-inflammatory and immunosuppressive agent that has been used in the treatment of various pulmonary diseases. 26 It potently inhibits the proliferation and production of cytokines and interleukins (such as IL-5, IL-6, tumor necrosis factor-α, prostaglandin, and cycloxygenase-2) and activation of T cells to reduce inflammation. 27,28 In addition, it reverses silicosis through its anti-fibrotic activity and inhibits mitogen-induced lymphocyte proliferation and antibody production by B cells in human myeloid leukemia cells through its immunosuppressive effects. 6,29 Kim et al. 4 showed that TET significantly reduced the levels of inflammatory cytokines, including interferon-λ, IL-1β, IL-6, and IL-8, induced by HCoV-OC43 infection in MRC-5 cells. The mechanism by which TET alleviates SARS-CoV-2-induced lung injury and inflammation needs further investigation. Combined with the multifunctional role of TET in treating severe lung damage, it is important to explore the clinical use of TET for the treatment of severe COVID-19.
Although it has been reported that there is no sex difference in the PK characteristics of TET in rats, 30 we found significant sex differences in the effects of TET in rat plasma ( Figure 5). The C max and AUC values of plasma TET in female rats were approximately twice as high as those in male rats at the same dosage and administration routes (i.v., p.o., and i.t.), which might be attributable to sex-related differences in drug-metabolizing enzymes in rats. Metabolic reaction phenotyping experiments showed that TET is metabolized mainly by CYP3A ( Figure S3). CYP levels in female rats are 10%-30% lower than those in male rats. 31,32 However, in human populations, variability in xenobiotic metabolism is predominantly related to intra-individual variation due to environmental exposure, rather than sex-dependent differences in CYP3A isoforms. 33 Therefore, in humans, TET metabolism is unlikely to differ by sex, which is why we used in vivo data and carried out an analysis based on the results from male rats.
Following oral administration, TET was slowly absorbed (T max = 6.7 h), with an oral bioavailability (F) value of 33.6%. The fraction of TET oral dose absorbed (F a × F g ) in rats (36.5%) was very close to that of F (33.6%), and the very low hepatic blood CL and extraction rate (3.26%) were consistent with the outcomes obtained from the metabolic stability experiment in RLM, suggesting that liver metabolism might not be the primary elimination pathway of TET. The low plasma concentration might not have been induced by hepatic metabolism. Our results suggest that the extrapolated human hepatic extraction rate from HLM (3.04%) was similar to that of rats; therefore, we speculated that the PK behavior of TET in humans might be similar to that of rats. After i.v. administration of TET in rats, V d (24.8 ± 0.952 L/kg) was much larger than the total extracellular fluid volume in rats (approximately 0.15 L/kg), 34 suggesting that TET is widely distributed in tissues and circulation after entering the bloodstream. 35,36 The plasma concentration of TET in the elimination phase decreased slowly and fluctuated, regardless of the route of administration, probably because of the slow release of TET from tissues into the blood.
At the beginning of the experiment after oral administration, the plasma concentration of TET was far below the EC 90 determined in the context of SARS-CoV-2 infection. Even with correction for the measured high R b/p (3.08) value, the concentration of TET in whole blood remained considerably below the EC 90 . To explore the pulmonary exposure levels of TET at the target sites, the tissue distribution of TET in rats was assessed. The biodistribution results in rats showed that the concentration of TET in major tissues was much higher than that in plasma, and exposure was extremely high in the liver, spleen, lung, kidney, and intestine. Considering the physiological characteristics of these tissues and the physicochemical properties of TET, the possible mechanisms of the high distribution of TET might involve: (1) active transporters located on the membrane of these tissues facilitating the distribution of TET against the concentration gradient 37-39 ; (2) TET sequestration in tissues rich in lysosomes through pH-driven lysosomal trapping, which could lead to enormous concentrations within lysosomes 40 ; or (3) TET entering monocytes/macrophage-rich tissues with circulating monocytes in the blood. 41 However, the mechanism needs further investigation. Regardless of the underlying mechanism, high pulmonary engagement with TET is beneficial for the treatment of lung diseases.
Although a high pulmonary distribution of TET is the desired outcome against COVID-19, extensive exposure in other tissues, especially in the liver, may pose a risk of toxicity. 42 Considering that administration by inhalation leads to higher pulmonary accumulation and lower liver accumulation, 43,44 we investigated the TET distribution profiles in rats after i.t. administration. The results showed that the K p value of the lungs was increased, but the K p values of the other tissues decreased significantly after i.t. administration compared with oral administration ( Table 1). Administration of TET by inhalation could help to achieve an effective concentration of TET in the lungs and reduce the risk of toxicity in other tissues.
In this study, in vivo functional readouts of TET were evaluated in a mouse-based SARS-CoV-2 infection model, while PK assessments were conducted in uninfected rats. An interspecies scaling approach based on body surface area (BSA) was used to estimate PK profiles in mice. Among the approaches for dose translation between laboratory animals, allometric scaling by BSA produces a better correlation between plasma volume/total circulating plasma protein than dosing based on either height or weight. 45 According to the BSA method, a dose of 60 mg/kg in mice was equivalent to a dose of approximately 30 mg/kg in rats. Therefore, in this study, in vivo efficacy was bridged with PK by dose translation.
Although a robust antiviral effect is obtained when the free concentration at the target site is above the in vitro unbound EC 90 value, the unbound plasma concentration is usually taken as a surrogate because the unbound plasma and unbound lung concentrations are assumed to be in equilibrium by rapid blood perfusion through the lung. 46 However, as TET has an extremely high tissue distribution, the use of plasma concentrations to evaluate its antiviral effect may severely underestimate its efficacy. By taking the highest unbound EC 90 value (worst-case scenario of 1.52 µg/ml) as an efficacy indicator, studies on tissue distribution showed that the unbound concentration in the lung could be above unbound EC 90 after repeated dosing of TET at 30 mg/kg through oral administration (from 0.43 µg/ml after a single dose to 2.28 µg/ml after multiple doses). This was confirmed by an in vivo efficacy study in mice receiving TET at doses above 60 mg/kg. Although, in this study, lung exposure after a single i.t. administration of 7 mg/kg was not effective (0.61 µg/ml), it has the potential to improve tissue specificity and reduce dose and toxicity risk after repeated i.t. administration due to its ability to yield higher lung exposure and lower exposure to other tissues compared to oral administration, and eventually found a new way for TET development.
Finally, the relationships between dose-route-target site exposures were investigated to anticipate human efficacy based on available PK profiles for TET repurposing. Regarding the clinical PK of TET, a reliable plasma C max of 67.3 ng/ml after a single oral administration of 100 mg TET tablet was identified. 16 Considering that K p,uu is a critical parameter to bridge PK and pharmacodynamics, as well as across species extrapolation, 47 the unbound lung concentration in humans after single oral dose of 100 mg TET would be approximately 0.24 µg/ml according to our present investigation. Currently, the clinical dosing regimen of TET for the treatment of silicosis in China is recommended as 60-100 mg/time, three times daily for 6 days, and 1 day off for 3 months. 5,48 Integrated with the tissue distribution results in rats after six consecutive days of administration, it is likely that the unbound lung concentration could be close to the effective exposure. However, our study also provides evidence that inhalation is a better route for reducing off-target toxicity. Another limitation of this study is that, owing to biosafety restrictions, all PK studies were conducted in normal animals, and PK studies in virus-infected mice have not been designed to obtain more direct lung exposure concentrations. This is a direction for future research.
In this study, we systemically confirmed the antiviral potency of TET in both in vitro and in vivo models. TET efficiently blocked SARS-CoV-2 infection in vitro and significantly alleviated virus-induced murine lung injury, suggesting that it might have dual mechanisms of both inhibiting viral replication and attenuating inflammatory response. The plasma or whole blood concentration of TET is not a proper surrogate because of its wide distribution and retention in the lungs. After a comparison of the biodistribution and pulmonary targeting ability of TET via different administration routes to the in vitro antiviral activity against SARS-CoV-2, a favorable biodistribution of TET after i.t. administration suggested that inhaled administration may be a more effective route than oral dosing to treat COVID-19. Finally, a clinical perspective of TET against SARS-CoV-2 is proposed. This work helps to better understand the biodistribution of TET in the respiratory tract under different administration routes, which will bridge the gap between in vitro activity and in vivo efficacy and facilitate the translational development of TET in the context of SARS-CoV-2 infection. The crucial pharmacodynamic and PK data obtained in this study provide useful references for further optimization of the therapeutic strategies of TET in treating COVID-19 and other diseases.

Cells and virus
Vero E6 cells were cultured in minimum Eagle's medium supplemented with 10% fetal bovine serum (FBS; Gibco Invitrogen); Huh7 and Calu-3 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS. SARS-CoV-2 (nCoV-2019BetaCoV/Wuhan/WIV04/2019 strain) was propagated in Vero E6 cells, and the virus stock was stored at −80 • C and titrated by an end-point dilution assay, as previously described. 21 All infection experiments were performed in a biosafety level-3 laboratory.

Chemicals and materials
TET (purity >99.2%; chemical structure shown in Figure  S1) was supplied by Conba Pharmaceutical Co., Ltd.

Animals
Eight to twelve-week-old SPF, female transgenic IFNAR −/− C57/BL6 mice were bred in the animal center of the Wuhan Institute of Virology, CAS (WIVA01202001). Healthy Sprague-Dawley rats (200-230 g) were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. The animals were housed in an appropriate environment and provided chow and water ad libitum. The rats were fasted for 12 h before oral administration of drug. All experiments were conducted at the Beijing Center for Drug Safety Evaluation after obtaining ethical approval (IACUC-DWZX-2021-662) from the Institutional Animal Care and Use Committee of the Center, which followed the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International.

Evaluation of TET against SARS-CoV-2 replication
The antiviral evaluation of the compounds in vitro was performed as previously described. 21 First, the cytotoxicity of TET on Vero E6, Huh7, and Calu-3 cells was detected using CCK8 kit (Beyotime, China). To determine the antiviral efficiency of the drug, monolayer cells cultured in a 48well cell-culture petri dish were pre-treated with different concentrations of TET for 1 h at 37 • C and incubated with SARS-CoV-2 at 0.05 or 0.01 MOI for 2 h. Then, the drugvirus mixture was washed and replaced with the fresh drug-containing medium. The supernatant of the infected cells was harvested at 24 h p.i. (Vero E6) or 48 h p.i. (Calu-3 and Huh7), and viral RNA levels in the supernatant were quantified using qRT-PCR.

Exploring the mechanism of TET in inhibition of SARS-CoV-2 infection
To define the step of SARS-CoV-2 infection targeted by TET, a time-of-addition assay was performed as previously described. 21 Vero E6 cells were treated with TET (5 µM) during entry, post-entry, or the entire infection process (full-time) (MOI = 0.05). At 16 h p.i., the virus yield in the supernatant was quantified by qRT-PCR. The expression of viral NP in the infected cells was analyzed by western blotting and IFA. 48 To investigate the impact of TET on viral intracellular trafficking, a colocalization analysis of virions with endosomes was carried out as previously described. 49 Briefly, a monolayer of Vero E6 cells cultured in the 35-mm glass-bottom culture dishes was pretreated with TET (10 µM) or DMSO and incubated with SARS-CoV-2 (MOI = 5) at 4 • C for 1 h. Then, the supernatant was replaced with the drug-containing medium. And the cells were cultured at 37 • C and then fixed with 4% (w/v) paraformaldehyde at different time points (15,30,45, and 60 min p.i.). Anti-NP, anti-EEA1, or anti-LAMP1 antibodies were used for immunofluorescence staining of the viral particles, EEA1 + EEs, and LAMP1 + endosomes/lysosomes, respectively. Fluorescence images were captured using a two-photon microscope (A1RMP, Nikon). The proportion of endosome (green) colocalized virus particles (yellow) to all virus particles (red) in the cells (n > 150 cells) was quantified using ImageJ (Colocation Threshold plugin). Statistical significance was assessed using one-way analysis of variance.

Antiviral assessment of TET in vivo
The antiviral efficiency of TET against SARS-CoV-2 infection in vivo was tested in AdV5-hACE2-transduced, aged 8-12 weeks SPF, male IFNAR −/− C57/BL6 mice, as previously described. 17 In brief, the mice were challenged with 1 × 10 6 TCID 50 of SARS-CoV-2 intranasally at 5 days post-transduced with 2.5 × 10 8 TCID 50 of AdV5-hACE2, administered intranasally. The infected mice were intraperitoneally injected with vehicle or TET (90, 60, and 30 mg/kg) daily for five consecutive days. The infected mice were observed daily and sacrificed at 5 days p.i. The lungs were subjected to viral load analysis using qRT-PCR, titrated using an end-point dilution assay, and histopathological analysis.

Pharmacokinetic study in rats via different administration routes
Preclinical PK studies of TET were conducted in rats via i.v., i.t., and p.o. administration (n = 6 for each group, male:female = 1:1). One group of rats was administered an i.v. injection (dissolved in 1.2% glacial acetic acid) at 5 mg/kg. Another group received intragastrically administered 30 mg/kg TET suspended in 0.5% carboxymethyl cellulose sodium (CMC-Na) solution. The other two groups of rats were administered by i.t. administration (simulating drug delivery by inhalation) at 7 and 14 mg/kg using the RM-003 liquid atomizer (dissolved in 22.5 mg/ml aspartic acid solution). Blood samples were collected from the jugular vein into heparin-anticoagulant tubes when the rats were anesthetized with isoflurane at 0, 0.033 (i.v. dosing only), 0.083, 0.25, 0.5, 1, 2, 4,6,8,12,24,36,48,72,96, and 120 h according to the pilot study results. Blood samples (approximately 100 µl) were centrifuged at 2500 × g at 4 • C for 10 min to harvest plasma within 1 h and were kept at −40 • C for analysis.

Tissue distribution study in rats via p.o. and i.t
Biodistribution characteristics in various organs of the TET were compared via p.o. and i.t. routes. Forty-five male Sprague-Dawley rats were randomly divided into 15 groups (n = 3 for each group). Five groups of rats received a single oral administration of TET (30 mg/kg) suspended in 0.5% CMC-Na solution, five groups received repeated oral administration of TET (30 mg/kg) daily for six consecutive days, and the other five groups received a single i.t. injection of TET (7 mg/kg), as described in Section 4.7. For each route of administration, one group of rats was euthanized at the desired time points (2, 6, 24, 144, and 240 h). The major organs and tissues, including the heart, liver, spleen, lung, kidney, brain, stomach, intestine, testis, ovary, uterus, adipose, muscle, and plasma, were immediately harvested, and all the tissues were thoroughly rinsed in physiological saline. All the samples were weighed and stored at −40 • C for analysis.

Protein binding and blood/plasma partitioning
The protein binding rates of TET in plasma, liver microsome incubate (0.5 mg/ml protein), tissue homogenates, and 2% FBS were determined using the equilibrium dialysis method according to the instructions of the RED device (details in Supporting Information).
The R b/p of TET was determined using freshly collected rat blood 4 h after oral administration of 30 mg/kg TET (n = 3) to protect blood cells from damage by any organic solvent introduced during the dissolution of TET. The obtained blood samples were divided into two parts: one part was immediately centrifuged for 10 min at 2500 × g to harvest the plasma, and the other part was sonicated for 15 min to break the blood cells. Each sample was matrix matched and subjected to protein precipitation. The concentration of TET in the blood and plasma was determined using LC-MS/MS. The R b/p ratio for TET was calculated as the ratio of the TET concentration in the blood to that in the plasma.

4.10
Metabolic stability in liver microsomes TET (1 µM, 1% ACN) was incubated in RLM or HLM (0.5 mg/ml), diluted in 100 mM PBS (pH 7.4) containing MgCl 2 (3 mM). After pre-incubation for 5 min, the reaction was initiated by adding NADPH (final concentration of 1 mM) at 37 • C. Periodic aliquots of the incubation mixture were removed at 0, 5, 15, 30, and 60 min and quenched with ACN. After centrifugation, the supernatant was collected and analyzed for the depletion by LC-MS/MS. Negative control incubations in the absence of NADPH and positive control incubations in the presence of cocktailed CYP probes were performed simultaneously. Intrinsic clearance and hepatic clearance were calculated based on a well-stirred model. 50

CYP reaction phenotyping in recombinant human CYP enzymes
The optimized experimental conditions for CYP reaction phenotyping followed the above metabolic stability studies, with linear depletion obtained after incubation for 30 min.
TET (1 µM) was incubated with a panel of recombinant human CYP enzymes (CYP1A2, CYP2A6, CYP2B6,  CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2,  CYP3A4, CYP3A5, and CYP4F2, 20 pmol/ml each) in 100 mM PBS (pH 7.4) containing NADPH (1 mM) and MgCl 2 (3 mM) at 37 • C for 30 min (n = 3). A negative control group using microtomes infected with the control baculovirus was used simultaneously. The reaction was then quenched with ACN. Following centrifugation, the supernatant was analyzed using LC-MS/MS. The remaining percentage of TET in the individual rCYP isoforms was calculated by comparing the concentrations before and after incubation.
Bioavailability (F) was calculated using Equation (1): The tissue partition ratio (K p ) of each tissue was calculated using Equation (2): The unbound fraction of TET in plasma and tissue homogenates (f u,x ) was calculated using Equation (3) The unbound fraction in tissues was adjusted by the fold of the dilution factor (D), as shown in Equation (4) 51 : The unbound fraction was used to calculate the unbound drug concentration in the plasma and tissues.
The contribution of each rCYP was calculated using the TNR approach reported by Rodrigues. 52 The relative contribution of the CYP isoforms to TET metabolism was estimated using Equation (5) The normalized rate (NR) was calculated as the reaction rate multiplied by the mean specific content of the corresponding CYP in the native HLM for each rCYP, which was expressed as pmol/min/mg microsomal protein. 52 The TET depletion half-life in microsomal incubates and apparent CL int were calculated according to a well-stirred model. The natural log of concentration versus time was fitted using linear regression, and the slope (k) was converted to T 1/2 values, where T 1/2 = −0.693/k. The CL int was estimated using Equation (6). 53 CL int = 0.693 in vitro 1∕2 × volume of incubation (μl) amount of microsomal protein in incubation (mg) × 45 mg microsomes g liver × g liver kg body weight (6) The CL h,blood was calculated according to Equation (7), using a hepatic blood flow (Q) value of 55.2 ml/min/kg for rats, and 20.7 ml/min/kg for humans. 34 The f u,p , f u,mic , and R b/p were obtained from measured values.
CL h = × u, p ∕ ∕ × CL int ∕ u,mic + u, p ∕ ∕ × CL int ∕ u, mic The fraction of absorbed oral dose (F a × F g ) was estimated using Equation (8): The appropriate statistical analysis was performed for each experiment. In the statistical analyses, t-tests were used to compare the results of the control and treated groups. The statistical methods and statistical significance are described in the figure legends.

C O N F L I C T O F I N T E R E S T
The authors declare they have no conflicts of interests.

D ATA AVA I L A B I L I T Y S TAT E M E N T
All data are available from the corresponding authors upon request.

E T H I C S S TAT E M E N T
All the experiments were conducted in Beijing Center for Drug Safety Evaluation after obtaining an ethical approval (IACUC-DWZX-2021-662) from the Institutional Animal Care and Use Committee of the Centre, which followed the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).